This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-MH-107: Targets for Drug Discovery for Mental Disorders. Amphetamine acts as a Molecular Stent in the Dopamine Transporter By introducing the term 'molecular stent'in connection with amphetamine and the dopamine transporter, we are intending to introduce a new line of thinking into the general body of literature on drug discovery for mental disorders. We attempt through this term to frame another approach to the molecular explanation of how amphetamine and its close relative, methamphetamine, might work. We know that AMPH and METH act at human dopamine transporter (hDAT), which is the primary molecular target for these drugs. Dopamine transporters are critical to dopaminergic signaling in reward pathways, and therefore are central to substance abuse and addiction. With the long-term objective to study the molecular mechanisms underlying drug abuse and addiction as a basis for drug discovery, we propose a new concept suggested by new experimental evidence that amphetamines lodge in transporter and open a pathway for ions that is not ordinarily there, and that the currents thus generated depolarize dopaminergic neurons, increase their excitability, and help explain the response to these addictive drugs. We propose to study extensively, in a concentrated, well manned, two-year project, DA, AMPH, and METH uptake and induced currents in hDAT-expressing Xenopus oocytes using simultaneous radio-labeled uptake and two-microelectrode voltage-clamp techniques. At saturating concentrations, AMPH and METH induce larger currents than DA, and, once exposed to these drugs, a baseline holding current persistently shifted the membrane potential in the depolarizing direction, which a long period of buffer perfusion could not wash out. The magnitude of baseline shifts depends on the concentration, the duration of exposure, and the chemical structure of the drugs, with AMPH more potent than METH. AMPH or METH exposure also reduced subsequent DA-induced currents, suggesting that the number of active transporters is compromised after exposure to AMPH or METH. RTI-55, a cocaine analogue hDAT inhibitor, eliminated the entire depolarizing current and restored the original baseline, suggesting the binding of AMPH or METH does not remove hDAT from the membrane;rather, the transporter is stabilized by the drugs to a ligand-gated, channel-like state causing a leak current through the transporter we describe as a molecular stent. An application of high positive membrane potentials only partially removed AMPH from the transporter, implying AMPH binds tightly and the stent persists long after the drug is removed. The constitutive leak current through the AMPH- or METH-exposed transporter is likely to be crucial to understanding the molecular mechanism of drug addiction: in this model, dopaminergic neurons would be depolarized by the leak, therefore becoming more excitable and more likely to release transmitter. We propose that the formation of a molecular stent by AMPH or METH within hDAT is an important feature of the persistent effects of these drugs after withdrawal. PUBLIC HEALTH RELEVANCE: The proposal 'Amphetamine acts as a Molecular Stent in the Dopamine Transporter'addresses the specific problem of amphetamine addiction, and introduces drug- development strategies for detoxification of drug abusers. The central hypothesis rests on data suggesting that amphetamines become lodged in the dopamine transporter, which introduces a pathway for Na+ ions that persistently depolarize dopaminergic neurons, releasing dopamine inappropriately. Drug development relies on chemical similarities between amphetamine, which introduces a stent, and dopamine, which does not.